In order to obtain over-expressed target protein to use the obtained target protein for medicine, an industry, or the like, various expression systems such as a microorganism expression system, a plant expression system, a yeast expression system, an insect cell expression system, an animal cell expression system, and the like, have been used. Among them, the microorganism expression system, which is the most easily used system, has been developed as expression systems suitable for various applications and commercialized.
However, the microorganism expression system has several limiting factors. The main limiting factor is that since protein expression and modification mechanism (glycosylation, phosphorylation, amidization) of the microorganism are different from that of an animal cell, even though the same gene is expressed in the microorganism expression system, a structure or feature of the expressed protein is not completely the same as that of the original protein. Therefore, in the case of producing recombinant protein using the microorganism expression system, since modification after synthesis was hardly generated, inactivation of the produced protein or a significant difference in functions is not generated, but modified protein or protein having a partial difference in the structure is frequently expressed. In addition, a production process of the recombinant protein using the microorganism expression system has difficulty in that a secondary contaminant removal process should be performed due to contamination of the microorganism, endotoxin contamination of the microorganism, or the like.
On the other hand, although the animal cell expression system is the most suitable system for animal protein expression, the animal cell expression system has a high production cost due to low expression efficiency of the recombinant protein, and an operating process of the animal cell is difficult, as compared to the microorganism expression system, such that it is not easy to industrialize the animal cell expression. As a currently used industrial animal cell line, there are Chinese Hamster Ovary (CHO) cells, Baby Hamster Kidney (BHK) cells, myeloma cells, or the like. The target foreign protein may be expressed by transfecting an expression vector including the corresponding gene in these animal cell lines.
In the case in which various protein modification mechanisms including glycosylation are maintained in the animal cell and protein is secreted in a culture medium, obtaining and purifying processes of the protein may be easily performed. Most of the animal cells necessarily require a composite additive such as serum protein, or the like during a culturing process, but since the CHO cell may be cultured in a medium to which serum and protein are not added, the CHO cell may be used as the most suitable host cell for recombinant protein expression. In addition, the CHO cell has advantages in that various researches into the CHO cells have been conducted, such that the feature thereof has been well known, a growth rate is rapid, and suspension culture for mass culture may be performed.
Generally, in the case of allowing a transgene to be expressed in animal cells, the transgene and a vector having a marker are simultaneously transfected, and the transfected cells are cultured in selective media and selected. However, in most cases, an expression frequency thereof is significantly low. One of the reasons is that these transgenes should be integrated into a chromosome of the host cell in the animal cell unlike the microorganism system. Further, even though stable transfectants in which the transgene is stably integrated into the chromosome of the host cell are selected, it is difficult to predict an expression amount thereof. The reason is that an integration position of the gene is different in each cell, and an expression pattern is different according to the integration position. Therefore, the number of transgenes and the expression amount of the transgene integrated into the animal cell do not have a clear correlation therebetween (Grindley et al., 1987, Trends Genet. 3, 16-22; Kucherlapati et al., 1984, Crit. Rev. Biochem. 16, 349-381; Palmiter et al., 1986, Annu. Rev. Genet. 20, 465-499). In most cases, gene expression in the animal cell is suppressed by DNA bases around the integration position, such that even in the case of stably integrated transgenes, the expression is often expressed at a significantly low level (Eissenberg et al., 1991, Trends Genet. 7, 335-340; Palmiter et al., 1986, Annu. Rev. Genet. 20, 465-499).
Availability of a DNA factor for protecting transgene expression from the gene position-specific effect as described above has been reported in various systems. As the above-mentioned DNA factor, an insulator factor, a nuclear matrix attachment region (hereinafter, referred to as “MAR”), a scaffold attachment region (hereinafter, referred to as “SAR”), or the like, may be used. Although operation mechanisms thereof have not been clarified, when the DNA factors are included in transgene constructs, they induce gene expression regardless of the integration position, and the expression amount is determined by the copy number of gene (McKnight, R. A. et al., 1992, Proc. Natl. Acad. US. 89, 6943-6947). Kalos et al. have combined the MAR element of the human apolipoprotein B gene with a minimal promoter transgene construct and induced gene expression in animal cells to increase expression of the transcript by about 200 times (Kalos et al., 1995, Mol. Cell. Biol. 15, 198-207). Similarly, it has been reported that the MAR element of the chicken lysozyme A gene, the SAR element of human interferon β, and the like confer transgene expression in vertebrates regardless of an integration position in chromosome of host cells (Eissenberg et al., 1991, Trends Genet. 7, 335-340; Klehr et al., 1991, Biochemistry 30, 1264-1270). However, an attempt to substantially increase protein production in cell lines using a combination of the specific β globin MAR element, the specific MAR/SAR element, or the specific interferon β SAR element as described above or the case in which an industrial profit was identified have not yet been reported.
This information disclosed in the present background art is only to improve understanding of a background of the present invention. Therefore, information on the prior art that is already known by those skilled in the art to which the present invention pertains may not be included.